Serum Protease Research Articles

The levels of serine proteases in colon tissue interstitial fluid and serum serve as an indicator of colorectal cancer progression.

The proteins in tissue interstitial fluids (TIFs) can spread into the blood and have been proposed as an ideal material to find blood biomarkers. The colon TIFs were collected from 8-, 13-, 18-, and 22-week ApcMin/+, a typical mouse model of colorectal cancer (CRC), and wild-type mice. iTRAQ-based quantification proteomics was conducted to survey the TIF proteins whose abundance appeared to depend on tumor progression. A total of 46 proteins that exhibited consecutive changes in abundance were identified, including six serine proteases, chymotrypsin-like elastase 1 (CELA1), chymotrypsin-like elastase 2A (CEL2A), chymopasin, chymotrypsinogen B (CTRB1), trypsin 2 (TRY2), and trypsin 4 (TRY4). The observed increases in the abundance of serine proteases were supported in another quantitative evaluation of the individual colon TIFs using a multiple reaction monitor (MRM) assay. Importantly, the increases in the abundance of serine proteases were also verified in the corresponding sera. The quantitative verification of the serine proteases was further extended to the clinical sera, revealing significantly higher levels of CELA1, CEL2A, CTRL/chymopasin, and TRY2 in CRC patients. The receiver operating characteristic analysis illustrated that the combination of CELA1 and CTRL reached the best diagnostic performance, with 90.0% sensitivity and 80.0% specificity. Thus, the quantitative target analysis demonstrated that some serine proteases are indicative of CRC progression.

Cellular Uptake of IgG Using Collagen-Like Cell-Penetrating Peptides.

Cell-penetrating peptides (CPPs) are attractive tools for delivering macromolecules that have poor membrane permeability, such as antibodies, into cells. However, the major drawback of conventional CPPs is their instability in bodily fluids. We previously reported a novel CPP employing a collagen-like triple-helical structure that exhibited remarkable resistance against serum proteases. Herein, we report the delivery of full-length immunoglobulin G (IgG) antibody into cells using a triple-helical CPP. The CPP was conjugated to IgG via a one-pot reaction using 2-iminothiolane as a crosslinking reagent. The triple-helical CPP was less prone to being aggregated and neutralized by serum than was octaarginine, a conventional CPP. However, most of the conjugates were found to be entrapped in endosomes.

Online 2D-LC-MS/MS assay to quantify therapeutic protein in human serum in the presence of pre-existing antidrug antibodies.

The formation of antidrug antibodies (ADA) can interfere with the accurate quantitation of therapeutic proteins, leading to significantly underestimated drug concentrations and confounded pharmacokinetic (PK) data interpretation. Although highly desirable, development of ADA-tolerant bioanalytical methods enabling unbiased measurement of both free and ADA-bound drug presents a considerable challenge. We report herein the development and validation of a robust LC-MS assay capable of quantifying therapeutic protein immunoglobulin A1 protease (IgAP) in human serum in the presence of pre-existing anti-IgAP antibodies. The procedure included sodium dodecyl sulfate (SDS) denaturation and chemical reduction of serum proteins to dissociate ADA-drug bindings, followed by tryptic digestion of protein pellets and subsequent LC-MS analysis of the surrogate IgAP peptide using stable isotope labeled peptide internal standard. Substantial enhancements in the sensitivity and selectivity were achieved by the combination of online two-dimensional reversed-phase LC (2D-LC) operated in high and low pH buffers, respectively, for efficient enrichment and quantitation of the surrogate peptide by multiple-reaction monitoring (MRM) mass spectrometry. Unlike ligand-binding assay, our method is not prone to interferences from ADA, allowing accurate and precise measurement of the IgAP in the range of 0.05 to 10 μg/mL in 25 μL of human serum with a wide range of anti-IgAP antibody levels. The intra- and inter-run precision (coefficient of variation (CV%)) was within 11.5% and 10.5%, respectively, and the bias was within ±7.1% for all quality control (QC) concentrations. With little modification, the described method can readily be applicable to the quantitation of other biotherapeutic proteins in the ADA-positive clinical matrices.

Abstract 1237: Antitumor studies of an E2f1 promoter sequence binding peptide - penetratin conjugate as a molecule targeting E2f in prostate cancer

Abstract BACKGROUND: Mutation or inactivation of the retinoblastoma protein is frequently involved in prostate cancer tumorigenesis resulting in overexpression/deregulation of E2F activity. E2F1-3a overexpression induces genes involved in DNA synthesis and leads to abnormal cellular proliferation, tumor growth, and invasion. Therefore, inhibiting the overexpression of one or more activating E2Fs is a recognized target in cancer therapeutics. In our previous studies we showed that a novel penetratin conjugated 7-mer peptide (PEP) bound tightly to an immobilized consensus E2F1 promoter sequence, was cytotoxic at low micro molar concentrations to many malignant cell lines and as the PEP was unstable in serum, the PEP was encapsulated in PEGylated liposomes and treatment of tumor xenografts of small cell lung cancer H-69 and DU145 tumors propagated in mice caused tumor regression. OBJECTIVE: To determine the antitumor activity and stability of two different modified penetratin peptides: D-Arg PEP (substituting L-Arginine with D-Arginine in the peptide sequence) and N-acetylated as well as C-methylated PEP analog. METHODS: DU145 (prostate cancer) and H196 (small cell lung cancer) cells were used. To compare the efficacy of the peptides, we tested the IC50s of peptides at different time points using the MTS assay. Drug combination experiment results were analyzed using the combination index (CI) method. Peptide conformational studies were carried out using the Amber 12 suite of biomolecular simulation programs. RESULTS: Molecular simulation studies showed that the D-Arg PEP secondary structure is more stable than the L-Arg peptide structure in water. D-Arg PEP was more potent compared to L-Arg PEP, and it was also found to be more resistant to degradation by serum proteases than the L-form. The other modified form, N-acetylated, C-methylated PEP was marginally more effective than the unmodified PEP. Drug combination studies showed that the D-Arg PEP in combination with docetaxel, caused synergistic cytotoxicity against DU 145 cells. Our findings validate D-Arg peptide, an inhibitor of E2F1and 3a transcription, as a drug candidate for targeted molecular therapy of prostate cancers with elevated levels of activated E2F’s. Studies in progress are evaluating the combination of the PEGylated liposome encapsulated D-Arg PEP in combination with docetaxel against DU145 xenografts and against primary prostate cancer cells. Supported in part by a grant from the Lung Cancer Research Foundation. Citation Format: Tazeem Shaik, Nitu Bansal, Nadine Johnson Farley, John Kerrigan, Olga Garbuzenko, Tamara Minko, Emine Abali, Zoltan Szekely, Kathleen Scotto, Debabrata Banerjee, Joseph Bertino. Antitumor studies of an E2f1 promoter sequence binding peptide - penetratin conjugate as a molecule targeting E2f in prostate cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1237. doi:10.1158/1538-7445.AM2015-1237

Protein profiles in mucosal and systemic compartments in response to Vibrio cholerae in a mouse pulmonary infection model

We have recently shown that a mouse lung infection model resulting in acute pneumonia could be used for evaluating the protective immunity induced by mucosal vaccines against Vibrio cholerae. In order to gain insight and better understanding of the pathogenicity of V. cholerae infection, we identified and compared proteins induced by V. cholerae in nasal washes, bronchoalveolar lavages (BAL), and sera. Intranasal administration of V. cholerae increased the concentration of total proteins in nasal washes and BAL fluids, but not in sera. LTQ-Orbitrap hybrid Fourier transform mass spectrometry showed that cytoskeletal proteins, protease inhibitors and anti-inflammatory mediators were present in nasal washes from uninfected mice. The distinctly expressed proteins in nasal washes in response to V. cholerae mainly consisted of protease inhibitors, anti-inflammatory proteins, and anti-microbial proteins. A number of protease inhibitors and anti-inflammatory proteins were selectively expressed in BAL fluids from V. cholerae-infected mice, while cytoskeletal proteins and heat shock proteins were mainly observed in BAL fluids from uninfected mice. A large number of serum complements, protease inhibitors, and acute phase proteins were expressed in V. cholerae-infected mice. Collectively, these results suggest that intranasal administration of V. cholerae leading to acute pneumonia elicited alterations of protein profiles associated with immune homeostasis and host protection in both the mucosal and systemic compartments.

α2-Macroglobulin Can Crosslink Multiple Plasmodium falciparum Erythrocyte Membrane Protein 1 (PfEMP1) Molecules and May Facilitate Adhesion of Parasitized Erythrocytes.

Rosetting, the adhesion of Plasmodium falciparum-infected erythrocytes to uninfected erythrocytes, involves clonal variants of the parasite protein P. falciparum erythrocyte membrane protein 1 (PfEMP1) and soluble serum factors. While rosetting is a well-known phenotypic marker of parasites associated with severe malaria, the reason for this association remains unclear, as do the molecular details of the interaction between the infected erythrocyte (IE) and the adhering erythrocytes. Here, we identify for the first time a single serum factor, the abundant serum protease inhibitor α2-macroglobulin (α2M), which is both required and sufficient for rosetting mediated by the PfEMP1 protein HB3VAR06 and some other rosette-mediating PfEMP1 proteins. We map the α2M binding site to the C terminal end of HB3VAR06, and demonstrate that α2M can bind at least four HB3VAR06 proteins, plausibly augmenting their combined avidity for host receptors. IgM has previously been identified as a rosette-facilitating soluble factor that acts in a similar way, but it cannot induce rosetting on its own. This is in contrast to α2M and probably due to the more limited cross-linking potential of IgM. Nevertheless, we show that IgM works synergistically with α2M and markedly lowers the concentration of α2M required for rosetting. Finally, HB3VAR06+ IEs share the capacity to bind α2M with subsets of genotypically distinct P. falciparum isolates forming rosettes in vitro and of patient parasite isolates ex vivo. Together, our results are evidence that P. falciparum parasites exploit α2M (and IgM) to expand the repertoire of host receptors available for PfEMP1-mediated IE adhesion, such as the erythrocyte carbohydrate moieties that lead to formation of rosettes. It is likely that this mechanism also affects IE adhesion to receptors on vascular endothelium. The study opens opportunities for broad-ranging immunological interventions targeting the α2M—(and IgM-) binding domains of PfEMP1, which would be independent of the host receptor specificity of clinically important PfEMP1 antigens.

Mechanistic investigation of the preclinical pharmacokinetics and interspecies scaling of PF-05231023, a fibroblast growth factor 21-antibody protein conjugate.

PF-05231023, a long-acting fibroblast growth factor 21 (FGF21) analog, was generated by covalently conjugating two engineered [des-His1, Ala129Cys]FGF21 molecules to a nontargeting human IgG1 κ scaffold. The pharmacokinetics (PK) of PF-05231023 after i.v. and s.c. administration was evaluated in rats and monkeys using two enzyme-linked immunosorbent assays with high specificity for biologically relevant intact N termini (NT) and C termini (CT) of FGF21. Intact CT of FGF21 displayed approximately 5-fold faster systemic plasma clearance (CL), an approximately 2-fold lower steady-state volume of distribution, and at least 5-fold lower bioavailability compared with NT. In vitro serum stability studies in monkeys and humans suggested that the principal CL mechanism for PF-05231023 was degradation by serum proteases. Direct scaling of in vitro serum degradation rates for intact CT of FGF21 underestimated in vivo CL 5-fold, 1.4-fold, and 2-fold in rats, monkeys, and humans, respectively. The reduced steady-state volume of distribution and the bioavailability for intact CT relative to NT in rats and monkeys were compatible with proteolytic degradation occurring outside the plasma compartment via an unidentified mechanism. Human CL and PK profiles for intact NT and CT of FGF21 were well predicted using monkey single-species allometric and Dedrick scaling. Physiologically based pharmacokinetic models incorporating serum stability data and an extravascular extraction term based on differential bioavailability of intact NT and CT of FGF21 in monkeys improved accuracy of human PK predictions relative to Dedrick scaling. Mechanistic physiologically based pharmacokinetic models of this nature may be highly valuable for predicting human PK of fusion proteins, synthetically conjugated proteins, and other complex biologics.

Intact stable isotope labeled plasma proteins from the SILAC-labeled HepG2 secretome.

The plasma proteome remains an attractive biospecimen for MS-based biomarker discovery studies. The success of these efforts relies on the continued development of quantitative MS-based proteomics approaches. Herein we report the use of the SILAC-labeled HepG2 secretome as a source for stable isotope labeled plasma proteins for quantitative LC-MS/MS measurements. The HepG2 liver cancer cell line secretes the major plasma proteins including serum albumin, apolipoproteins, protease inhibitors, coagulation factors, and transporters that represent some of the most abundant proteins in plasma. The SILAC-labeled HepG2 secretome was collected, spiked into human plasma (1:1 total protein), and then processed for LC-MS/MS analysis. A total of 62 and 56 plasma proteins were quantified (heavy:light (H/L) peptide pairs) from undepleted and depleted (serum albumin and IgG), respectively, with log2 H/L = ± 6. Major plasma proteins quantified included albumin, apolipoproteins (e.g., APOA1, APOA2, APOA4, APOB, APOC3, APOE, APOH, and APOM), protease inhibitors (e.g., A2M and SERPINs), coagulation factors (e.g., Factor V, Factor X, fibrinogen), and transport proteins (e.g., TTR). The average log2 H/L values for shared plasma proteins in both undepleted and depleted plasma samples were 0.43 and 0.44, respectively. This work further expands the SILAC strategy into MS-based biomarker discovery of clinical biospecimens.

Isolated Defect of Intestinal Iron Absorption in Siblings of Iron Deficiency Anemia

Iron deficiency anemia (IDA) is the commonest type of anemia all over the world. It is easily treatable with oral iron therapy, but a minority of patients does remain unresponsive. This may be related to inappropriate compliance, manifest or occult blood loss, worm infestations, helicobacter infection, known malabsorption syndromes or paroxysmal nocturnal hemoglobinuria (PNH). We observed six such patients with non-responsive recurrent classical IDA who did not show any of the above conditions on specific investigations. Iron tolerance test (as an index of oral iron absorption) was performed on these patients. All of them showed negligible or no iron absorption, suggesting a defect in intestinal oral iron absorption as the cause of non-responsive IDA. This contention was supported by a dramatic response to parenteral iron therapy in all of them. Since the tests for known malabsorption syndromes were negative, it would be assumed that these subjects had an isolated defect in oral iron absorption that has been only scantily reported in the literature. Five of the six patients were siblings of two families. This raises the possibility of the defect in iron absorption to have a genetic connotation. Pending the complete work up for genetic studies, including the gene mutation for membrane bound serum protease type 6 (TMPRSS6) in these subjects and their families, this presentation is a short communication to expose and share our small experience of what looks to be an isolated defect in intestinal iron absorption.

Abstract 4939: Detection and quantification of enzymatically active tissue prostate-specific antigen in vivo

Abstract Prostate-Specific Antigen (PSA) is a 237 amino acid, 33 kDa, extracellular serine protease expressed in prostate epithelial tissue. In the normal prostate, high concentrations of PSA are stored in the prostatic ductal network. However, cancer leads to the disruption of normal tissue architecture and subsequent leakage of PSA into the tissue interstitium and circulation. Enzymatically active PSA is only present in prostate tissue and at other sites of prostate cancer growth. In circulation, active PSA immediately forms complexes with the serum protease inhibitor alpha-1-antichymotrypsin (ACT), while the inactive forms remains “free”. Traditional assays relied on testing for the presence of total PSA (mostly inactive and complexed) and do not provide information regarding the amount of enzymatically active PSA, a more biologically relevant tumor biomarker. To fill this gap, we developed a novel near infrared (NIR) fluorescent agent, designed to detect active PSA with no detection of inactive or complexed PSA. This agent, PSA750 FAST, contains a PSA-cleavable peptide sequence labeled with NIR fluorophores (ex/em 750/770 nm) and coupled to a pharmacokinetic modifier designed to improve its plasma availability. In its native state, the agent is nearly completely optically quenched (> 95%); and only becomes fluorescent upon enzymatic cleavage with active PSA, yielding a 300-1800 fold increase in signal as compared to the signal obtained using inactive or complexed PSA. Proteolytic cleavage was also selective for PSA over a large panel of enzymes, including Kallikrein 1, Cathepsin B, MMP-9, MMP-12, MMP-13, uPA, chymotrypsin and thrombin. We hypothesized that the enzymatic activity of PSA could be non-invasively and quantitatively monitored in 3D using Fluorescence Molecular Tomography (FMT), a powerful near-infrared imaging modality that enables 3D quantitative determination of fluorochrome distribution in tissues of live small animals. LNCaP (PSA positive) and PC3 (PSA negative) cells were implanted in the chest area of male nude mice. Real time imaging was performed after systemic administration of PSA750 FAST when tumors reached the desired size. Our results demonstrated a significantly higher fluorescent signal in LNCaP tumors as compared the adjoining muscle (34.1 +/- 3.8 nM versus 7.2 +/- 0.6 nM, p=0.0043). Likewise, fluorescence concentration in tumors was significantly higher in LNCaP as compared to PC3 tumors (34.1 +/- 3.8 nM vs 17.14 +/- 3.48 nM, p=0.0191; 12.99 +/- 3.7 vs 2.8 +/- 0.8 pmoles, p=0.0233). This is the first report that demonstrates the feasibility of non-invasive, real-time, molecular imaging of active PSA in vivo in a relevant tumor model. These findings demonstrate the effectiveness of this agent in conjunction with optical imaging as a functional platform for molecular imaging in the field of prostate cancer. Citation Format: Jeffrey D. Peterson, Guojie Ho, Jeffrey Morin, Jeannine Delaney, Wael Yared, Milind Rajopadhye, Sylvie C. Kossodo. Detection and quantification of enzymatically active tissue prostate-specific antigen in vivo. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4939. doi:10.1158/1538-7445.AM2014-4939

Abstract 699: Mesenchymal stem cells (MSC) as cell-based vectors for PSA-activated proaerolysin to sites of prostate cancer

Abstract Mesenchymal stem cells (MSC) traffic to sites of inflammation, such as those commonly found at sites of prostate and other cancers. Using multi-parameter flow cytometry, our group has shown that MSCs represent 0.01-1.1% of the total cells present at sites of primary prostate cancer (PCa). Allogeneic MSCs have been safely administered to >1000 patients in over 350 clinical trials worldwide for a variety of diseases. Coupled with their ability to evade the immune system, this suggests that allogeneic MSCs can be used as cell-based delivery vectors for anti-cancer agents. Towards this goal, a method has been developed to load MSCs with PLGA microparticles (MP) encapsulating PSA-activated proaerolysin. Proaerolysin is a highly potent (low pM) bacterial pore-forming toxin that selectively kills PSA-expressing cells in a proliferation-independent manner, which is critical due to the low proliferative index of PCa. Proaerolysin has been modified using site-directed mutagenesis to replace the wildtype activation domain with a PSA cleavage sequence. Though prodrug delivery will be enriched at cancer sites using MSC ‘Trojan horses’, entrapment in non-malignant tissue, such as the lung, following systemic administration is expected and must be addressed; here, using a prodrug strategy. Importantly, enzymatically active PSA is only present in the prostate and at sites of PCa, including metastases. Circulating PSA is inactive due to covalent binding to serum protease inhibitors. Therefore, toxicity to non-target tissues will be minimized through both selective delivery and prodrug activation. The prodrug is released from the MPs in a controlled manner over at least a 1 week period in vitro. Hemolysis assays in the presence of MP-conditioned supernatant demonstrated that MP fabrication does not neutralize drug toxicity. Furthermore, incubation of LNCaP (PSA+) and PC3 (PSA-) cells with MP-conditioned supernatant demonstrates selective toxicity to PSA-expressing cells at low nM concentrations. MP internalization by MSCs has been confirmed using both flow cytometry and confocal microscopy. Chitosan-modification of the MPs permitted increased loading of MSCs (100 ug/mL of MPs). For long-term development, preclinical studies such as these have raised the question of tumor homing efficiency in humans. Therefore, an ongoing FDA-approved first-in-man pre-prostatectomy clinical trial to quantify the number of systemically-delivered allogeneic MSCs that traffic to sites of primary PCa has been initiated. BEAMing (digital PCR) technology will be used to accurately quantify donor MSCs based on differential SNP profiles (detection threshold: ≥0.01%). Data from this trial will be used to determine the amount of prodrug-loaded MPs necessary to deliver per MSC to achieve a therapeutic effect. This data will subsequently be used in ongoing animal studies to model clinical relevance in efficacy studies prior to further translation. Citation Format: W. Nathaniel Brennen, Oren Levy, Sudhir Ranganath, Michael Schweizer, Marc Rosen, Sandrine Billet, Neil Bhowmick, Samuel Denmeade, Jeffrey Karp, John Isaacs. Mesenchymal stem cells (MSC) as cell-based vectors for PSA-activated proaerolysin to sites of prostate cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 699. doi:10.1158/1538-7445.AM2014-699

Serum and salivary matrix metalloproteinases, neutrophil elastase, myeloperoxidase in patients with chronic or aggressive periodontitis.

Salivary, serum matrix metalloproteinase-8 (MMP-8), tissue inhibitor of matrix metalloproteinases-1 (TIMP-1), neutrophil elastase (NE), and myeloperoxidase (MPO) levels were investigated in generalized chronic periodontitis (GCP), generalized aggressive periodontitis (GAgP), and healthy groups. Whole-mouth clinical periodontal measurements were recorded. Salivary, serum concentrations of MMP-8, MPO, TIMP-1, and NE were determined by immunofluorometric assay or ELISA in 18 patients with GCP, 23 patients with GAgP, 18 individuals with healthy periodontium. Patients in the GAgP group were younger than the other groups (p<0.05). The study groups were similar in gender, smoking status. Plaque index was higher in GCP than GAgP group (p<0.05). Biochemical data were similar in periodontitis groups. Salivary, serum MPO, and salivary NE concentrations were higher; TIMP-1 concentrations were lower in the periodontitis groups than the controls (p<0.05). The present data support a close relationship between salivary, serum protease content and clinical periodontal parameters in patients with generalized periodontitis.

Inter-α-Inhibitor Blocks Epithelial Sodium Channel Activation and Decreases Nasal Potential Differences in ΔF508 Mice

Increased activity of lung epithelial sodium channels (ENaCs) contributes to the pathophysiology of cystic fibrosis (CF) by increasing the rate of epithelial lining fluid reabsorption. Inter-α-inhibitor (IαI), a serum protease inhibitor, may decrease ENaC activity by preventing its cleavage by serine proteases. High concentrations of IαI were detected in the bronchoalveolar lavage fluid (BALF) of children with CF and lower airway diseases. IαI decreased amiloride-sensitive (IENaC) but not cAMP-activated Cl(-) currents across confluent monolayers of rat ATII, and mouse nasal epithelial cells grew in primary culture by 45 and 25%, respectively. Changes in IENaC by IαI in ATII cells were accompanied by increased levels of uncleaved (immature) surface α-ENaC. IαI increased airway surface liquid depth overlying murine nasal epithelial cells to the same extent as amiloride, consistent with ENaC inhibition. Incubation of lung slices from C57BL/6, those lacking phenylalanine at position 508 (∆F508), or CF transmembrane conductance regulator knockout mice with IαI for 3 hours decreased the open probability of their ENaC channels by 50%. ∆F508 mice had considerably higher levels the amiloride-sensitive fractions of ENaC nasal potential difference (ENaC-NPD) than wild-type littermates and only background levels of IαI in their BALF. A single intranasal instillation of IαI decreased their ENaC-NPD 24 hours later by 25%. In conclusion, we show that IαI is present in the BALF of children with CF, is an effective inhibitor of ENaC proteolysis, and decreases ENaC activity in lung epithelial cells of ∆F508 mice.

Efficacy of a metalloproteinase inhibitor in spinal cord injured dogs.

Matrix metalloproteinase-9 is elevated within the acutely injured murine spinal cord and blockade of this early proteolytic activity with GM6001, a broad-spectrum matrix metalloproteinase inhibitor, results in improved recovery after spinal cord injury. As matrix metalloproteinase-9 is likewise acutely elevated in dogs with naturally occurring spinal cord injuries, we evaluated efficacy of GM6001 solubilized in dimethyl sulfoxide in this second species. Safety and pharmacokinetic studies were conducted in naïve dogs. After confirming safety, subsequent pharmacokinetic analyses demonstrated that a 100 mg/kg subcutaneous dose of GM6001 resulted in plasma concentrations that peaked shortly after administration and were sustained for at least 4 days at levels that produced robust in vitro inhibition of matrix metalloproteinase-9. A randomized, blinded, placebo-controlled study was then conducted to assess efficacy of GM6001 given within 48 hours of spinal cord injury. Dogs were enrolled in 3 groups: GM6001 dissolved in dimethyl sulfoxide (n = 35), dimethyl sulfoxide (n = 37), or saline (n = 41). Matrix metalloproteinase activity was increased in the serum of injured dogs and GM6001 reduced this serum protease activity compared to the other two groups. To assess recovery, dogs were a priori stratified into a severely injured group and a mild-to-moderate injured group, using a Modified Frankel Scale. The Texas Spinal Cord Injury Score was then used to assess long-term motor/sensory function. In dogs with severe spinal cord injuries, those treated with saline had a mean motor score of 2 (95% CI 0–4.0) that was significantly (P<0.05; generalized linear model) less than the estimated mean motor score for dogs receiving dimethyl sulfoxide (mean, 5; 95% CI 2.0–8.0) or GM6001 (mean, 5; 95% CI 2.0–8.0). As there was no independent effect of GM6001, we attribute improved neurological outcomes to dimethyl sulfoxide, a pleotropic agent that may target diverse secondary pathogenic events that emerge in the acutely injured cord.

Are Serum Alpha-1-Antitrypsin and Alpha-2-Macroglobulin Levels Related with Tumor Histology or Stage of the Lung Cancer?.

In certain types of cancer, increased serum protease inhibitor levels were identified and clues showing that, serum levels of protease inhibitor could be used in the assessment of prognosis and dissemination of tumor were obtained. We aimed to reveal whether serum alpha-1-antitrypsin (AAT) and alpha-2-macroglobulin (AMG) levels of patients with lung cancer are related with histological type, clinical stage, and advance of cancer. The patients enrolled in the study were the ones with a histopathologic diagnosis of primary lung cancer and they were not administered any treatment before. Levels of serum AAT and AMG were analyzed at the moment of diagnosis. A total of 47 patients were enrolled in the study – 42 males and 5 females. The average age was 58.9. Tumor cell type was non-small cell lung carcinoma (NSCLC) in 36 and small cell lung carcinoma in 11 patients. There was a negative correlation between serum AAT level and age (p= 0.21 r=-0.339). No correlation was identified between serum AMG level and age (p= 0.138 r= 0.222). There was no significant relation between histological types of lung cancer and serums AAT and AMG (p= 0.807, p= 0.411 respectively). AAT and AMG levels were not different in limited and extensive small cell lung carcinoma (p= 0.201 and p= 0.465). AAT and AMG levels based on stages were not found to be different in NSCLC (p= 0.646 and p=0.650). AAT and AMG levels did not vary based on distant metastasis or local spread in NSCLC (p= 0.371 and p= 0.676). Serum alpha-1-antitrypsin and alpha-2-macroglobulin levels in patients with lung cancer do not vary based on histological types, stage of the disease, local, distant and lymphatic spread of the tumor.

Hydroxyproline Substitutions Stabilize Non-Glycosylated Drosocin Against Serum Proteases Without Challenging its Antibacterial Activity

The increasing incidence of multi- and pan-resistant pathogens demands novel compounds to fight Grampositive and especially Gram-negative bacteria. Among the currently investigated compound classes, antimicrobial peptides (AMPs) inhibiting specific bacterial targets appear especially promising for systemic therapy of infections, although unmodified linear peptides are typically rapidly degraded by serum proteases. Proline-rich AMPs have been heavily investigated in recent years due to their low toxicity and proven in vivo efficacy. Here, we report novel unglycosylated drosocin analogs with extended half-life in mouse serum and improved activity against Gram-negative pathogens Escherichia coli and Klebsiella pneumoniae. Substituting proline (Pro) residues in positions 3, 5, 10, and 14 with trans-4-hydroxy-Lproline ((t)Hyp) improved the antibacterial activity, whereas substitution of Pro-16 reduced the activity. Drosocin analogs with (t)Hyp in positions 3 and 5 were also four to eight times more stable in mouse serum than the unmodified analog. The new compounds were not toxic against human HeLa, HEK293, and HepG2 cell lines and showed no hemolytic activity against human erythrocytes at peptide concentrations of at least 600 µg/mL.

A new method to assay protease based on amyloid misfolding: application to prostate cancer diagnosis using a panel of proteases biomarkers.

This paper reports a sensitive method with electrochemical technique to detect various proteases, which can be used for the diagnosis of prostate cancer. For the proposed assay method, the working electrode is modified with the peptide probes for the target proteases. These probes contain the substrate sequence of target proteases, as well as the seed peptide sequence that can accelerate the misfolding of amyloid-beta. If there are proteases in the test solution, after protease cleavage of the substrate peptides, the distal seed peptide will be removed from the electrode surface. So, in the absence of proteases, the seed peptides can initiate and accelerate amyloid-beta misfolding on the electrode surface. Consequently, the formed aggregates strongly block the electron transfer of the in-solution electroactive species with the electrode, resulting in suppressed signal readout. Nevertheless, in the presence of proteases, enzyme cleavage may lead to greatly mitigated protein misfolding and evident signal enhancement. Since the contrast in signal readout between the two cases can be amplified by using the protein misfolding step, high sensitivity suitable for direct detection of proteases in serum can be achieved. These results may suggest the feasibility of our new method for the detection of a panel of proteases in offering detailed diagnosis of prostate cancer and a better treatment of the cancer.

18F-FDG PET/CT Findings Preceded Elevation of Serum Proteinase 3 Antineutrophil Cytoplasmic Antibodies in Wegener Granulomatosis

A 67-year-old woman underwent F-FDG PET/CT after developing a fever of unknown origin. PET/CT revealed intensive FDG uptake at the nasal and lung lesions. On the laboratory data, serum myeloperoxidase antineutrophil cytoplasmic antibodies (ANCA) titer was elevated, although serum directed against proteinase 3 (PR3) ANCA titer was within normal limits. One month after treatment, follow-up PET/CT revealed decreased FDG uptake at the lesions. One year later, serum PR3-ANCA titer elevated, which finally led to a diagnosis of Wegener granulomatosis (WG). WG lesions may be detected earlier by FDG PET/CT than by serum PR3-ANCA titers.

Using antisense technology to develop a novel therapy for α-1 antitrypsin deficient (AATD) liver disease and to model AATD lung disease.

Alpha-1 antitrypsin (AAT) is a serum protease inhibitor that belongs to the serpin superfamily. Mutations in AAT are associated with α-1 antitrypsin deficiency (AATD), a rare genetic disease with two distinct manifestations: AATD lung disease and AATD liver disease. AATD lung disease is caused by loss-of-function of AAT and can be treated with plasma-derived AAT. AATD liver disease is due to the aggregation and retention of mutant AAT protein in the liver; the only treatment available for AATD liver disease is liver transplantation. Here we demonstrate that antisense oligonucleotides (ASOs) targeting human AAT efficiently reduce levels of both short and long human AAT transcript in vitro and in transgenic mice, providing a novel therapy for AATD liver disease. In addition, ASO-mediated depletion of mouse AAT may offer a useful animal model for the investigation of AATD lung disease.

From venoms to BBB shuttles: Synthesis and blood–brain barrier transport assessment of apamin and a nontoxic analog

Venoms are currently the focus of many drug discovery programs because they contain highly bioactive and selective components. Among them, apamin, a peptide found in bee venom, has received considerable attention because of its affinity for certain potassium channels and also because of its interesting structure and high stability to extreme pH and temperatures. Although apamin has long been claimed to cross the blood-brain barrier (BBB), only a few studies have been performed producing controversial results. In this article, it is shown that not only apamin is indeed able to penetrate the BBB in a cell-based model but also that an analog reported to be nontoxic passes through this barrier. Furthermore, the permeability values obtained, together with some evidence of an active transport mechanism and an amazing stability to serum proteases, make these peptides promising candidates for BBB shuttles.